The goal of this project is to elucidate the functions of mammalian H1 linker histones in gene regulation and development. Along with the four core histones (H2A, H2B, H3 and H4) that assemble DNA into nucleosomes, the H1 linker histones play key roles in organizing the structure of the chromatin fiber. Mammals express multiple, non-allelic, evolutionarily conserved H1 variants or subtypes that differ in their primary sequences and in their expression during development. These subtypes, each encoded by a single copy gene, provide additional levels of regulation of chromatin function. Although there has been substantial progress in understanding the roles of core histones in chromatin structure and function, current knowledge about H1's is quite limited. We propose to help fill this gap by elucidating the functions, mechanisms of action and subtype-specific activities of mammalian H1 linker histones in gene regulation and development. To investigate the functions of mammalian H1's in vivo, we have generated and characterized mouse embryonic stem cells and mice in which one or several H1 genes have been inactivated by gene targeting. We also have studied the functions of H1's with chromatin reconstituted in vitro. These studies have revealed unexpected roles and modes of action for H1 in helping to control DNA and histone methylation. The work also has uncovered important functional differences among the H1 subtypes. To gain a deeper understanding of the mechanisms by which H1 participates in gene regulation and development, we propose to pursue the following specific aims: (1) To elucidate the functions of H1 linker histones in regulating core histone post-translational modifications; (2) To understand the mechanisms by which certain H1 subtypes help control DNA methylation; (3) To determine the roles played by H1's in red blood cell development. The successful completion of the proposed work will lead to new insights into mechanisms of gene regulation and a deeper understanding of the functional significance of the diversity present in the mammalian H1 family. It will also begin to define the roles of this major constituent of chromatin in the well-defined process of red blood cell development.